Inoculation Effect of Methanotrophs on Rhizoremediation Performance and Methane Emission in Diesel-Contaminated Soil

During the rhizoremediation of diesel-contaminated soil, methane (CH4), a representative greenhouse gas, is emitted as a result of anaerobic metabolism of diesel. The application of methantrophs is one of solutions for the mitigation CH4 emissions during the rhizoremediation of diesel-contaminated soil. In this study, CH4-oxidizing rhizobacteria, Methylocystis sp. JHTF4 and Methyloversatilis sp. JHM8, were isolated from rhizosphere soils of tall fescue and maize, respectively. The maximum CH4 oxidation rates for the strains JHTF4 and JHM8 were 65.8 and 33.8 mmol·g-DCW-1·h-1, respectively. The isolates JHTF4 and JHM8 couldn't degrade diesel. The inoculation of the isolate JHTF4 or JHM8 significantly enhanced diesel removal during rhizoremediation of diesel-contaminated soil planted with maize for 63 days. Diesel removal in the tall fescue-planting soil was enhanced by inoculating the isolates until 50 days, while there was no significant difference in removal efficiency regardless of inoculation at day 63. In both the maize and tall fescue planting soils, the CH4 oxidation potentials of the inoculated soils were significantly higher than the potentials of the non-inoculated soils. In addition, the gene copy numbers of pmoA, responsible for CH4 oxidation, in the inoculated soils were significantly higher than those in the non-inoculated soils. The gene copy numbers ratio of pmoA to 16S rDNA (the ratio of methanotrophs to total bacteria) in soil increased during rhizoremediation. These results indicate that the inoculation of Methylocystis sp. JHTF4 and Methyloversatilis sp. JHM8, is a promising strategy to minimize CH4 emissions during the rhizoremediation of diesel-contaminated soil using maize or tall fescue.


Identification of the Isolates
To identify the strain JHTF4 and JHM8, its colony on the NMS agar plate was picked and added into 100 μl of distilled water using a sterile loop. After then, the cell suspension was heated at 95 o C and centrifuged to obtain its DNA. After PCR using the DNA template and 340F and 805R primer targeting 16S rRNA [21,22], the sequences of the PCR products were analyzed by Macrogen (Republic of Korea). The resulting sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) developed by the National Center for Biotechnology Information (NCBI). The sequences of the strain JHTF4 and JHM8 were deposited into the NCBI GenBank database under accession number MZ045833 and ON573373, respectively. A phylogenetic tree was constructed with the 16S rRNA sequences of the strain MZ045833 and ON573373, respectively. The strain JHTF4 and JHM8 was known as Methylocyctis sp. and Methyloversatilis sp. using the MEGA software (version 11, www.megasoftware.net) and the neighbor-joining algorithm.
To confirm whether each isolate was a pure culture, the following tests were conducted. Each culture broth cultivated in the NMS-CH 4 medium, where CH 4 was supplied as a sole carbon and energy source, was spread on an LB-agar plate to confirm whether heterotrophic bacteria were contaminated. In addition, after diluting the culture broth with sterile water, the diluted solution was spread on the NMS-agar plate, the plate was incubated in an aerobic jar containing 5% (v/v) CH 4 , at 30°C for 14 days. Ten colonies grown on the NMS-agar plate were randomly selected, and their 16S rRNA sequences were analyzed to determine whether they were identical to the nucleotide sequences of each isolate.

Characterization of CH 4 Oxidization by the Isolates
Each isolate was pre-grown in a 1.2-L serum bottle, containing 300 ml of NMS medium and 5% (v/v) CH 4 at 30°C for 10 days. Each 20 ml of the pre-cultured broth (OD 600 = 1.5) was transferred into a 600-ml serum bottle. CH 4 gas was injected to be final concentration of 1, 5, 10, 15, or 20% (v/v) in the headspace of each bottle. Using Henry's constant (20°C, 1 atm) for CH 4 , the total CH 4 amount in each bottle is corresponded to 250, 1248, 2496, 3743, or 4991 mmole/bottle, respectively [23]. Using Henry's constant for CH 4 , each CH 4 concentration in the liquid is calculated as 14, 69, 139, 208, and 277 μM, respectively [23]. The rates of CH 4 oxidation were calculated from the slopes of plots of CH 4 concentration versus time. The maximum CH 4 oxidation rate (V max ) and saturation constant (K m ) were determined using a Lineweaver-Burk plot [24]. Cell concentrations were determined using the relationship between the optical density measured at 600 nm and dry cell weight (DCW). The optical density was measured at 600 nm using a Libra S22 spectrophotometer (Biochrom, UK). Each experiment was performed in triplicate.

Effect of Root Exudate on CH 4 Oxidization by the Isolates
The effect of root exudate on CH 4 oxidation rates by the strains JHTF4 and JHM8 was evaluated as follows. The root exudate of tall fescue and maize was prepared in a similar manner to that described in a previous paper [25]. Two concentrations of root exudate were prepared for each plant: The total organic carbon (TOC) concentrations of the maize root exudate were 126 and 252 mg·l -1 , and the TOC concentrations of the tall fescue root exudate were 43, 85, and 213 mg·l -1 . The chemical compounds in the NMS medium were also added to each root exudate at the same concentrations as in the NMS medium to produce the root exudate medium. Eight mL of the strain JHTF4 or strain JHM8 pre-culture broth was added to a 600-ml serum bottle containing 12 ml of the root exudate medium. For the control, the pre-culture broth was added into the NMS medium. CH 4 gas was injected to be final concentration of 5% (v/v) in the headspace of each bottle. All experiments were performed at 30°C with 150 rpm in triplicate. The CH 4 oxidation rate for each experimental condition was evaluated in a same manner as described above.

Preparation of Pot Experiments
Rhizoremediation of diesel-contaminated soil was conducted on the rooftop of the New Engineering Building at Ewha Womans University, Seoul, Republic of Korea (37°57' N, 126°95' E). Soil, collected from the rooftop garden, was sieved with a 2-mm sieve to remove weeds and stones. The soil texture was loamy sand. After the soil was artificially contaminated with diesel at initial concentrations of 10,000 mg-diesel·kg-soil -1 , the contaminated soil was then placed for 1 week. The soil was mixed manually once a day for 1 week. Compost (Seokgang Green Fertilizer Inc., Republic of Korea) was added to the contaminated soil to be final concentration of 5 % (w/w) to provide N and P [20].
The strains JHTF4 and JHM8 were cultured in the NMS medium supplemented with 5 % CH 4 , and each 20 ml of the culture broth (OD 600 = 1.5) was added into 2 kg of the diesel-contaminated soil sample. A drainer and coarse sand were first placed on the bottom of each pot (diameter of 180 mm; height of 150 mm), and each inoculated soil with the strain JHTF4 or JHM8 was then added to each pot. As control, 2 kg of the contaminated soil without inoculation was added to a pot. Ten tall fescue or five maize seedlings were planted in each pot. The pot experiment was conducted in triplicate. The tall fescue seedlings were cultivated from seed for 45 days in a garden on the rooftop of the New Engineering Building at Ewha Womans University. Maize seedlings were purchased (Mojong 114, Republic of Korea), and cultivated for 45 days in the garden. The pot experiment was conducted on the rooftop of the New Engineering Building, Ewha Womans University for 63 days (May 24 to July 26, 2021). The pots were watered periodically to keep the plant from wilting during experiment.

Inoculation Effect of the Isolates JHTF4 and JHM8 on Diesel Removal and Soil CH 4 -Oxidation Potential
To evaluate the inoculation effect of the isolates JHTF4 and JHM8 on diesel removal and soil CH 4 -oxidation potential during the rhizoremediation of diesel-contaminated soil, soil samples were taken randomly from each pot on days 0, 35, and 63. To analyze the residual diesel concentrations, the collected soil was freeze-dried, and 3 g of each sample was consequently added to individual test tubes, after which 10 ml of hexane-acetone (1:1, v/v) solution was added as the solvent for extraction. The diesel concentration was measured using the same method and gas chromatograph (GC 6980N system, Agilent Technologies, USA) as described in a previous study [20].
To measure soil CH 4 oxidation potential, after air-drying the collected soils at room temperature, 2 g of each soil sample was added to a 600-ml serum bottle containing 12 ml of the NMS medium. The CH 4 oxidation activity was evaluated in the same manner as described in Section 2.1.
To monitor the pmoA gene abundance in the diesel contaminated soil, qPCR was performed. The 16S rRNA gene was quantitatively evaluated for total bacteria abundances using 340F and 805R primer sets [20,22]. The pmoA gene abundance was measured using A189f/mb661r [20,26]. The ratio of pmoA gene to 16S rRNA gene was calculated to evaluate the relative abundance of CH 4 -oxidizing bacteria to total bacteria.

Statistical Analysis
Microsoft Excel 2013 (Microsoft Co., USA) was employed to conduct t-tests and multiple comparisons with a pvalue of 0.05 used to indicate a significant difference.

Identification and CH 4 Oxidation Activity of the Isolates JHTF4 and JHM8
The isolates JHTF4 and JHM8 from the rhizosphere soil samples planted with tall fescue and maize consortia were identified as Methylocystis sp. and Methyloversatilis sp., respectively (Fig. 1). Type I and type II methanotrophs have been differentiated by physiological characteristics [27]. Methylocystis spp., type II methanotrophs, assimilate formaldehyde by relatively less-productive pathway serine pathway [27]. Methylocystis spp. had been isolated form landfill cover soil, wetland, paddy soil, river, lake and so on [23,[28][29][30][31]. Methyloversatilis spp., type I methnotrphs, use the ribulose monophosphate pathway for formaldehyde assimilation [27]. Methyloversatilis spp. had been isolated from lake sediment, biofilm on steel-bentonite interphase, and so on [32,33]. Fig. 2 shows the specific CH 4 oxidation rates of Methylocystis sp. JHTF4 and Methyloversatilis sp. JHM8 under different CH 4 concentration (10,000-200,000 ppm). Both strains completely oxidized CH 4 without a lag phase under the CH 4 concentration of 50,000 ppm or less. They also completely oxidized 100,000 ppm CH 4 after short lag period of 4-8 h (data not shown). In the high concentration CH 4 condition of 150,000 ppm or more, CH 4 was oxidized after a lag phase of 8-12 h, and 60-90% of initial CH 4 was oxidized for 57 h. The specific CH 4 oxidation rates of strain JHTF4 were increased with increasing initial CH 4 concentration until 150,000 ppm CH 4 (208 μM in liquid phase), but the rate decreased at 277 μM CH 4 in the liquid ( Fig. 2A). However, the specific CH 4 oxidation rates of strain JHM8 were increased until 50,000 ppm CH 4 , and the rates were similar in the conditions from 50,000 to 200,000 ppm CH 4 ( Fig. 2A). At all CH 4 concentrations, the CH 4 oxidation rates of strain JHTF4 were superior to those of strain JHM8. Based on the Lineweaver-Burk plot (Fig. 2B), maximum CH 4 oxidation rate (V max ) and saturation constant (K m ) for strain JHTF4 were calculated as 65.8 mmol·g-DCW -1 ·h -1 and 41 μM, respectively. For strain JHM8, V max and K m were 33.8 mmol· g-DCW -1 ·h -1 and 48 μM, respectively. Methylocystis sp. M6 had isolated from a landfill cover soil, and its V max and K m were reported as 4.93 mmol·g-DCW -1 ·h -1 and 23 μM, respectively [23]. The CH 4 oxidation rates of strain JHTF4 and strain JHM8 were 13 and 7 times that of strain M6, respectively.

Effect of the Root Exudate on CH 4 Oxidation Activity
Rhizoremediation is a plant-assisted bioremediation using rhizobacteria, and has been applied in various ways to remediate soil or water contamination [34]. Rhizobacteria are influenced not only by physicochemical factors such as water content, pH, temperature, oxygen concentration, and nutrients, but also by root exudates secreted from plants [35]. Root exudates contain various organics and inorganics, and can act as a carbon source or growth  factor for rhizosphere bacteria [36][37][38]. Therefore, root exudates can affect rhizosphere bacterial metabolism and alter the biogeochemical cycling of carbon and nitrogen in soil ecosystems [39]. Maize and tall fescue are often used in rhizoremediation processes, and they grow well in harsh or various environments [6,7,20,40,41]. Fig. 3 shows the addition effects of the root exudates of maize and tall fescue on the CH 4 oxidation rates of strains JHTF4 and JHM8. Although the CH 4 oxidation rate of strain JHTF4 slightly increased by the addition of root exudates below 100-150 mg-TOC·L -1 , the CH 4 oxidation activity of strain JHTF4 was hardly affected by the addition of root exudates (Fig. 3A). The CH 4 oxidation rate of strain JHM8 was not affected by adding the root exudate of tall fescue, but it decreased with increasing the root exudate concentrations of maize (Fig. 3B). The addition effect of maize and tall fescue root exudates on the CH 4 oxidation rate of the CH 4 -oxidizing consortium was evaluated [25]. As a result, the CH 4 oxidation rate was slightly decreased by the exudate addition. It was considered that the activity of methanotrophs, which use C1 compound such and CH 4 and methanol as a substrate, was inhibited by the carbon source contained in the root exudates [25]. In this study, the CH 4 oxidation ability of the strain JHTF4 isolated from tall fescue rhizosphere was not inhibited by addition of maize root exudate as well as tall fescue. However, the CH 4 oxidation ability of the strain JHM8 isolated from maize rhizosphere was not affected by tall fescue root exudate, but it was inhibited by maize root exudate. In order to interpret these findings in detail, further study on qualitative and quantitative analysis of the components of maize and tall fescue exudates is needed.

Inoculation Effect of the Isolates JHTF4 and JHM8 on Diesel Degradation during Rhizoremediaiton
To evaluate the inoculation effect of the strains JHTF4 and JHM8 on the diesel degradation, the residual TPH concentrations during rhizoremediation using maize and tall fescue were measured (Fig. 4A). Both strain JHTF4 and strain JHM8 couldn't degrade diesel (data not shown). In the soils planted with maize or tall fescue, TPH removal efficiencies with inoculating the strain JHTF4 were significantly higher than those without the inoculation until 50 days (Fig. 4B). At the 65th day, the TPH removal in the soil planted with maize and inoculated with strain JHTF4 was significantly higher than that without inoculation (Fig. 4B). However, in the soil planted with tall fescue, there was no significant difference in removal efficiency regardless of inoculation (Fig. 4B). The inoculation effect of strain JHM 8 on TPH removal showed a similar trend to that of strain JHTF4 (Fig. 4C). Until day 50, TPH removal efficiency was improved by strain inoculation in maize and tall fescue planting soil, but at day 63, TPH removal was improved by strain inoculation only in soil planted with maize.
Diesel, which contains hydrocarbons with approximately 12-20 carbon atoms, and diesel is known to be degraded by microorganisms with the alkB gene or with the CYP153 enzyme in aerobic environment [9,42]. Some methylotrophs such as Methylophaga are known to have oil degrading ability [43], but the strains JHTF4 and JHM8 isolated in this study didn't have diesel-degrading ability. Therefore, the enhancement of diesel removal by inoculating the strains JHTF4 and JHM8 was not because they directly degraded diesel. Methanotrophs can utilize low-molecular hydrocarbon compounds and aromatic compounds as carbon sources [23,44,45]. Considering these capacity, the strains JHTF4 and JHM8 could improve diesel removal by playing a role in utilizing dieseldegrading intermediates that inhibit the activity of diesel-degrading bacteria during rhizoremediation process. Fig. 5 shows the inoculation effect of the strains JHTF4 and JHM8 on the CH 4 oxidation potential of rhizosphere soils planted with maize and tall fescue. In the soil planted with maize, the CH 4 oxidation potentials of the inoculated soil with the strains JHTF4 and JHM8 were significantly higher than the potentials of the noninoculated soil at 35th and 63th day (Fig. 5A). At 63th day, the CH 4 oxidation potentials with the inoculation of the strains JHTF4 and JHM8 were 1.16 (15.9 ± 0.19 mmol CH 4 ·g dry soil −1 ·h −1 ) and 1.07 (14.7 ± 0.07 mmol CH 4 ·g dry soil −1 ·h −1 ) times of the potential of the non-inoculated soil (13.70 ± 0.1 mmol CH 4 ·g dry soil −1 ·h −1 ), respectively. In the soil planted with tall fescue, the soil CH 4 oxidation potentials were also significantly enhanced by inoculating with the strains JHTF4 and JHM8 (Fig. 5B). At 63th day, the CH 4 oxidation potentials with the inoculation of the strains JHTF4 and JHM8 were 14.8 ± 0.07 and 14.8 ± 0.12 mmol CH 4 ·g dry soil −1 ·h −1 , while the CH 4 oxidation potential of the non-inoculated soil was 13.90 ± 0.06 mmol CH 4 ·g dry soil −1 ·h −1 . These results suggest that the CH 4 -oxidizing activities of the strains JHTF4 and JHM8 displayed in the soil system, resulting in an improvement  in soil CH 4 oxidation potentials. CH 4 is generally produced by methanogens in anaerobic zone of soil [46], but it is consumed by methanotrophs in aerobic zone of soil [47]. In addition, root-associated methanotrophs contribute in mitigating CH 4 emission from the wetlands and soils [7,20,25,47].

Inoculation Effect of the Isolates JHTF4 and JHM8 on CH 4 Oxidation Potential and pmoA Gene Dynamics in Soil
During rhizoremediation of diesel-contaminated soil, the inoculation effect of the strains JHTF4 and JHM8 on dynamics of the pmoA gene, which is responsible to CH 4 oxidation [7,20], is shown in Fig. 6. To compare total bacterial biomass in the soils without and with inoculation, 16S rRNA gene copy numbers were measured (Figs. 6A and 6D). 16S rRNA gene copy number weren't significantly different in all soils regardless of inoculation (3.1 × 10 6 -8.8 × 10 6 copy number•g-dry soil -1 in the maize planting soils and 3.3 × 10 6 -6.0 × 10 6 copy number•gdry soil -1 in the tall fescue planting soils). However, in both the maize and tall fescue planting soils, the pmoA gene copy numbers in the inoculated soils were significantly higher than those in the non-inoculated soils (Figs. 6B and 6E). In the inoculated soils, the copy number ratio of pmoA gene to 16S rRNA gene (pmoA/16S rRNA ratio) tended to increase during rhizoremedition. In the maize planting soils with the strains JHTF4 and JHM8, the pmoA/16S rRNA ratios increased 4.2 and 4.7 times, respectively, from 35th day to 63th day. In addition, the pmoA/ 16S rRNA ratios also increased 6.3 and 3.8 times in the tall fescue planting soils with the strains JHTF4 and JHM8, respectively. CH 4 oxidation rate increased with pmoA gene copy numbers in beech soil [48]. In addition, the pmoA gene copy number and CH 4 oxidation activity tended to be proportional to each other in ammonium sulfateadded soil [49]. Soil CH 4 oxidation potentials were closely associated with pmoA gene copy numbers during rhizoremediation of diesel-contaminated soil [7,20]. Based on the results in Figs. 5 and 6, it can be confirmed that seen that Methylocystis sp. JHTF 4 and Methyloversatilis sp. JHM8, inoculated into the soils planted with maize or tall fescue, exhibits CH 4 oxidation activity while surviving well in the soils.

Conclusion
Considering global climate change by increasing greenhouse gas emissions, the innovation of rhizoremediation technology is required to mitigate CH 4 emission during the biodegradation of petroleum hydrocarbons such as diesel and gasoline. In this study, two methanotrophs were isolated, and the inoculation effect of the isolates on diesel removal and soil CH 4 oxidation potential was investigated during the rhizoremediation of dieselcontaminated soil planted with tall fescue (F. arundinacea) or maize (Z. mays). The isolate JHTF4 from the tall fescue rhizosphere and the isolate JHM8 from the maize rhizosphere were identified as Methylocystis sp. and Methyloversatilis sp., respectively. The maximum CH 4 oxidation rates were 65.8 mmol·g-DCW -1 ·h -1 for the isolate JHTF4 and 33.8 mmol·g-DCW -1 ·h -1 for the isolate JHM8, which are superior to the rate for previously reported Methylocystis sp. When each isolate was added to diesel-contaminated soil planted with tall fescue or maize, the soil CH 4 oxidation potentials as well as diesel removal efficiencies were significantly higher than those of the noninoculated soils. Moreover, the pmoA gene copy numbers and the pmoA/16S rRNA ratios were also higher in the inoculated soils. Overall, these results suggest by inoculating Methylocystis sp. JHTF4 and Methyloversatilis sp. JHM8, not only the rhizoremediation performance can be improved, but also methane emissions can be reduced during the rhizoremediation of diesel-contaminated soil using tall fescue or maize. This study is the first report showing the possibility of reducing CH 4 emissions and improving remediation efficiency by applying a pure methanotroph. Further studies are required to characterize the relationship among the inoculated methanotroph, indigenous soil bacteria and plant roots for the satisfactory remediation efficiency and CH 4 mitigation during the rhizoremediation.